Ammonium Formate vs. Ammonium Acetate in HILIC

[M Farooq]

Hello,

Does ammonium formate offer a different selectivity as compared to ammonium acetate in HILIC mode?

Thanks.

[Andy Alpert]

Modestly, at most. What's more important is the pH. There are three considerations here:

1) An unbuffered acid tends to be a chaotrope, and chaotropes antagonize retention in HILIC. If you're at a pH above the pKa of the anion (3.75 for formate and 4.75 for acetate), then this won't be an issue. If you're at a pH well below the pKa, then a lot of the anion will be in the unbuffered acid form.

2) The pH also affects the charge of an analyte. If you're at a pH below 4, then carboxyl- groups will lose their (-) charge which will affect the overall polarity of an analyte. If the analyte doesn't have any basic groups, then uncharging a carboxyl- group will make it less polar and retention should decrease. If the analyte does have basic groups which can interact to some extent with the carboxyl- groups, then uncharging the carboxyl- groups makes the analyte more (+) charged. (+) charged analytes are the most polar of all. In this case, then, retention should increase. Formate is a better buffer than is acetate at pH's low enough for carboxyl- groups to lose (-) charge.

3) If the stationary phase material has significant (-) charge from silanols, then formate can buffer the mobile phase more effectively at pH's low enough for those to lose their charge. That would affect the retention of charged analytes, by decreasing either electrostatic attraction or repulsion.

[M Farooq]

Thank you for the insight. Do you why an unbuffered acid disrupts the water layer (i.e. why it behaves as a chaotropic material).

[Andy Alpert]

The immobilized layer of water is probably held in place by hydrogen bonds. Chaotropes disrupt those. As to why unbuffered acids are chaotropes: The short answer is that I don't know. Now, there is some tendency for hydrophilic interaction to decrease as one goes to a very high pH, and perhaps the same is true at low pH because of the abundance of the H3O+ ions. This is a falsifiable hypothesis but nobody has had any incentive to test it yet.

[Vlad Orlovsky]

The effect of ammonium formate or ammonium acetate is more pronounced in our mixed-mode columns, since we are exploring HILIC and ion-exchange mechanisms. pH of you buffer not only change ionization state/polarity of your analyte but also changes polarity and ion-exchange properties of the stationary phase (Obelisc N). You can even achieve change in order of elution when you go from ammonium formate pH 3 to ammonium acetate pH 5. You can use this tool to adjust selectivity of your separation.

[Jimi]

I would like to jump in here please.

Andy: Your comment about the effect of chaotropic additives is interesting - and it makes logical sense. However, the limited data I have seen on this showed a fairly small effect. Are there papers out there that show a significant effect. Perhaps you could list one or two, if not too much trouble.

On a different topic - but still in the realm of "HILIC Additives" - does anyone know if Citric Acid, or its salts, can be used in HILIC (i.e. are they soluble in the high organic). I've tried to look this up, but I don't see this one listed on either the "do use" or the "don't use" list (at least in the references I have on hand).

Thank You for any feedback.

[M_Farooq]

On a different topic - but still in the realm of "HILIC Additives" - does anyone know if Citric Acid, or its salts, can be used in HILIC (i.e. are they soluble in the high organic). I've tried to look this up, but I don't see this one listed on either the "do use" or the "don't use" list (at least in the references I have on hand).

Thank You for any feedback.

Any complexing acid, like citric acid, is going to be a troublesome additive. It will most likely give huge system peaks. Any additive which interacts too strongly with a stationary phase is perhaps going to be problematic.

[M_Farooq]

The effect of ammonium formate or ammonium acetate is more pronounced in our mixed-mode columns, since we are exploring HILIC and ion-exchange mechanisms. pH of you buffer not only change ionization state/polarity of your analyte but also changes polarity and ion-exchange properties of the stationary phase (Obelisc N). You can even achieve change in order of elution when you go from ammonium formate pH 3 to ammonium acetate pH 5. You can use this tool to adjust selectivity of your separation.

This is interesting. What is your Obelisc N column chemistry (in generic terms if you wish). Thanks.

[Vlad Orlovsky]

Here is surface chemistry:
http://www.sielc.com/Products_Obelisc.html

[M_Farooq]

Here is surface chemistry:
http://www.sielc.com/Products_Obelisc.html

Thanks. Just curious, if the positive charge is so close to the silica surface (as shown), how is this bonded phase stable with respect to hydrolysis or is it a cross linked polymer coating?

[Vlad Orlovsky]

There is no issue with hydrolysis coming from the stationary phase itself. Even in neutral non-buffered pH of the mobile phase pH inside pores is not going to dangerous levels.

[Andy Alpert]

This is a belated response to Jimi's request of Nov. 13th. I needed some time to look up references comparing retention in HILIC using unbuffered acids (presumably acting as chaotropes) with retention using salts as the additives. Refs:
1) T. Yoshida, Anal. Chem. 69 (1997) 3038: This paper compares retention of some peptide standards on a TSK Amide-80 column at high levels of organic solvent with the following alternative additives: 0.1% TFA; 0.1% formic acid; 0.1% acetic acid; no additive. Retention increases in that order. This also correlates with the decreasingly chaotropic nature of the additive. It's hard to distinguish this effect from the consequences of converting the basic residues - normally the most hydrophilic parts of a peptide - to less polar ion pairs. An experiment in which that factor was controlled would involve peptides with no basic groups. Anyway, Yoshida seems to have had a little trouble distinguishing HILIC from HIC, the abbreviation for hydrophobic interaction chromatography, since he states that the high concentration of nonvolatile salts in that mode would be incompatible with the chromatographic conditions at hand (he calls his conditions normal phase liquid chromatography). Also, comparing his results with my paper in 1990 that introduced HILIC, he only addresses my results obtained with an SCX column and ignores the results obtained with the neutral material PolyHYDROXYETHYL A (which some might regard as a competitor of Amide 80...)
2) T. Yoshida, J. Chromatogr. A 840 (1999) 1: Here Yoshida compares retention of peptides on an Amide-80 column and a diol column in the HILIC mode (which he still calls normal phase) using either 0.1% TFA or 0.1% triethylammonium trifluoroacetate as additives. Retention of peptides is greater using the salt (which would be less chaotropic than the unbuffered acid).

In response to your inquiry about whether or not citric acid or its salts might be soluble in HILIC mobile phases: Sure, if you provide a sufficiently hydrophobic anion as the counterion. I've published a number of papers recommending the use of triethylammonium phosphate as an additive in HILIC.

[Jimi]

Thanks Andy

One last follow up on this thread. When you advocate triethylammonium phosphate as a HILIC additive, is that purely due to its higher organic solubility, or also because the larger cation more effectively competes for the silanol (or other) sites.

Thanks

[Andy Alpert]

The higher organic solubility. That said, experience has shown that having a lot of phosphate in the mobile phase lets you do some things that you can't do as effectively with other anions. For example, in ERLIC, starting the chromatography with sodium methylphosphonate in the mobile phase results in heightened selectivity for retention of phosphopeptides. Subsequently running a gradient to 0.2 M TEAP elutes them all, even ones with 4 phosphates. If you tried that with a volatile salt such as ammonium formate, you'd have trouble eluting all of the peptides with 3 phosphates, to say nothing of the ones with 4.

Historical note: When I started using TEAP, it was because it was transparent at 220 nm. At that time I only had access to an absorbance detector.

[M_Farooq]

The higher organic solubility. That said, experience has shown that having a lot of phosphate in the mobile phase lets you do some things that you can't do as effectively with other anions. For example, in ERLIC, starting the chromatography with sodium methylphosphonate in the mobile phase results in heightened selectivity for retention of phosphopeptides. Subsequently running a gradient to 0.2 M TEAP elutes them all, even ones with 4 phosphates. If you tried that with a volatile salt such as ammonium formate, you'd have trouble eluting all of the peptides with 3 phosphates, to say nothing of the ones with 4.

Historical note: When I started using TEAP, it was because it was transparent at 220 nm. At that time I only had access to an absorbance detector.

That is interesting.
A heads-up notice to new HILIC users is that many siloxane bonded phases HILIC on silica will probably die soon with 200 mM buffer. In my opinion only polymeric coatings on silica can handle such high salt concentrations, which Dr. Alpert is very comfortable with.